The hepatoma cell lines stably expressing sodium taurocholate cotransporting polypeptide (NTCP), the receptor of hepatitis B virus (HBV) infection, serve as important infection models for studying viral biology and drug discovery. However, the efficiency of infection greatly varies. In this study, we studied the effects and potential mechanisms of Matrigel® hESC-qualified (M-hq), a biological basement membrane matrix commonly used in cell culture, on promotion HBV in vitro infection in HepG2-NTCP cells. For the first time, our findings demonstrate that M-hq could enhance the infection efficiency of cell culture-derived HBV with no impact on the cell viability, the HBV transcription and response to antiviral treatments. The infection enhancement is reproducible and is suggested to occur at HBV attachment step. Our study suggests that this novel system is applicable for studying HBV biology and new drugs.

The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to public health. Human breast milk is a complex nutritional composition to nourish infants and protect them from different kinds of infectious diseases including COVID-19. Here, we identified that lactoferrin (LF), mucin1 (MUC1), and α-lactalbumin (α-LA) from human breast milk inhibit SARS-CoV-2 infection using a SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). In addition, LF and MUC1 inhibited multiple steps including viral attachment, entry, and postentry replication, whereas α-LA inhibited viral attachment and entry. Importantly, LF, MUC1, and α-LA possess potent antiviral activities toward variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), and B.1.617.1 (kappa). Taken together, our study provides evidence that human breast milk components (LF, MUC1, and α-LA) are promising antiviral and potential therapeutic candidates warranting further development for treating COVID-19. Keywords: Biological sciences; Microbiology; Viral microbiology.

Despite the presence of hepatitis B virus (HBV) in the human breastmilk of mothers infected with HBV, it has been shown that breastfeeding does not increase the risk of mother-to-child transmission (MTCT) of HBV. We tested the hypothesis that human breastmilk may contain active components that bind to HBV and inhibit the infectivity of HBV. The results show that human whey significantly inhibited the binding of the hepatitis B surface antigen (HBsAg) to its antibodies in competitive inhibition immunoassays. The far-western blotting showed that HBsAg bound to a protein of 80 kD in human whey, which was identified as lactoferrin by mass spectrometry. Competitive inhibition immunoassays further demonstrated that both human lactoferrin and bovine lactoferrin bound to HBsAg. Human whey, human lactoferrin, and bovine lactoferrin each significantly inhibited the infectivity of HBV in vitro. Our results indicate that human breastmilk can bind to HBsAg and inhibit the infectivity of HBV, and the active component is lactoferrin. The findings may explain the reason that breastfeeding has no additional risk for MTCT of HBV, although human breastmilk contains HBV. Our study provides experimental evidence that HBV-infected mothers should be encouraged to breastfeed their infants.

Members of the tripartite motif (TRIM) protein family strongly induced by interferons (IFNs) are parts of the innate immune system with antiviral activity. However, it is still unclear which TRIMs could play important roles in hepatitis B virus (HBV) inhibition. Here, we identified that TRIM56 expression responded in IFN-treated HepG2-NTCP cells and HBV-infected liver tissues, which was a potent IFN-inducible inhibitor of HBV replication. Mechanistically, TRIM56 suppressed HBV replication via its Ring and C-terminal domain. C-terminal domain was essential for TRIM56 translocating from cytoplasm to nucleus during HBV infection. Further analysis revealed that TRIM56's Ring domain targeted IκBα for ubiquitination. This modification induced phosphorylation of p65, which subsequently inhibited HBV core promoter activity, resulting in the inhibition of HBV replication. The p65 was found to be necessary for NF-κB signal pathway to inhibit HBV replication. We verified our findings using HepG2-NTCP and primary human hepatocytes. Our findings reveal that TRIM56 is a critical antiviral immune effector and exerts an anti-HBV activity via NF-κB signal pathway, which is essential for inhibiting transcription of HBV covalently closed circular DNA.

Neural tube defects (NTDs) are a group of common and severe congenital malformations. The PI3K-AKT signalling pathway plays a crucial role in the neural tube development. There is limited evidence concerning any possible association between aberrant methylation in PI3K-AKT signalling pathway genes and NTDs. Therefore, we aimed to investigate potential associations between aberrant methylation of PI3K-AKT pathway genes and NTDs. Methylation studies of PI3K-AKT pathway genes utilizing microarray genome-methylation data derived from neural tissues of ten NTD cases and eight non-malformed controls were performed. Targeted DNA methylation analysis was subsequently performed in an independent cohort of 73 NTD cases and 32 controls to validate the methylation levels of identified genes. siRNAs were used to pull-down the target genes in human embryonic stem cells (hESCs) to examine the effects of the aberrant expression of target genes on neural cells. As a result, 321 differentially hypermethylated CpG sites in the promoter regions of 30 PI3K-AKT pathway genes were identified in the microarray data. In target methylation analysis, CHRM1, FGF19, and ITGA7 were confirmed to be significantly hypermethylated in NTD cases and were associated with increased risk for NTDs. The down-regulation of FGF19, CHRM1, and ITGA7 impaired the formation of rosette-like cell aggregates. The down-regulation of those three genes affected the expression of PAX6, SOX2 and MAP2, implying their influence on the differentiation of neural cells. This study for the first time reported that hypermethylation of PI3K-AKT pathway genes such as CHRM1, FGF19, and ITGA7 is associated with human NTDs. Keywords: Neural tube defects,; PI3K-AKT signalling pathway; methylation.

Here, we present a protocol to characterize the antiviral ability of a protein of interest to SARS-CoV-2 infection in cultured cells, using MUC1 as an example. We use SARS-CoV-2 ΔN trVLP system, which utilizes transcription and replication-competent SARS-CoV-2 virus-like particles lacking nucleocapsid gene. We describe the optimized procedure to analyze protein interference of viral attachment and entry into cells, and RT-qPCR-based quantification of viral infection. The protocol can be applied to characterize more antiviral candidates and clarify their functioning stage.